A large number of Gram-negative bacteria naturally produce extracellular membrane vesicles (MVs). These vesicles are derived from the outer membrane (OM) and contain OM and periplasmic proteins. MVs are an important component of bacterial virulence as they serve as trafficking vehicles for bacterial toxins to eukaryotic cells. In addition to trafficking toxins, our laboratory recently discovered that the opportunistic pathogen Pseudomonas aeruginosa utilizes MVs to traffic the cell-cell signaling molecule PQS between P. aeruginosa cells. Since PQS signaling is required for virulence, MVs are a critical component of P. aeruginosa pathogenicity. Although the roles of MVs in pathogenesis are clear, the molecular mechanism of MV formation is not currently understood. The goal of this proposal is to determine the molecular mechanism of MV formation in P. aeruginosa. To this end, a molecular model for MV formation has been developed, and three specific aims are proposed to test this model. These specific aims propose genetic, biochemical, and physiological techniques to exact a role for PQS (specific aim 1), lipopolysaccharide (specific aim 2), and pg-associated OM proteins (specific aim 3) in MV formation, with the ultimate goal of precisely defining the mechanism of MV formation. Due to their role in toxin and signal trafficking, understanding how MVs are produced will provide new insight into P. aeruginosa signaling and disease. Ultimately, understanding the molecular mechanism of MV formation may provide a novel target for antimicrobial development through development of technology to control MV production. Bacterial communication within a population is critical for pathogenicity of many bacteria, including the bacterium Pseudomonas aeruginosa. The goal of this project is to provide a fundamental understanding of how P. aeruginosa communicates, with the ultimate goal of devising ways to disrupt this communication.